title: The effect of ligand binding on the galactokinase activity of yeast Gal1p and its ability to activate transcription.
abstract: The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Sellick
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jowitt
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom. Electronic address: richard.reece@manchester.ac.uk.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Reece
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism*; D005695:Galactosephosphates / Q000235:genetics / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics; D008024:Ligands; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: C099289:Gal3 protein, S cerevisiae; D005695:Galactosephosphates; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C029973:galactose-1-phosphate; D000255:Adenosine Triphosphate; C524219:GAL1 protein, S cerevisiae; D005686:Galactokinase